Ripa lysis buffer

Western blots were performed to determine the protein expression levels of CD24, CD133 and EpCAM in the liver tissues. Briefly, protein lysates were extracted from the liver tissues using RIPA Lysis Buffer (Aspen, Canada, USA) supplemented with the protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was determined by BCA protein kit (Aspen, Canada, USA). The protein samples were separated with 10% SDS-PAGE and transferred to PVDF membrane. Then, the membrane were incubated with the primary antibodies overnight at 4 °C, followed by the corresponding horseradish peroxidase conjugated secondary antibodies at room temperature for 30 min. Finally, the membrane was developed with the enhanced chemiluminescence (ECL) substrate solution kit (Aspen, Canada, USA) for 1 min. The targeted protein bands were analyzed using with AlphaEaseFC software for gray scale value. GAPDH was used as the internal control. Primary antibodies used in this study are listed in Table 2.Table 2

Detailed information for antibodies used for western blot

Antibody	Species	Manufacturer	Category no	Dilution ratio
GAPDH	Rabbit	Abcam	ab9485	1:10,000
CD24	Rabbit	Abcam	ab179821	1:500
CD133	Rabbit	Cell Signaling Technology	64,326	1:500
EpCAM	Rabbit	Abcam	ab223582	1:1000
Wnt3a	Rabbit	Abcam	ab219412	1:500
β-catenin	Rabbit	Abcam	ab32572	1:500

Canine LAA tissues were lysed with RIPA Lysis Buffer (ASPEN, USA) supplemented with complete protease and phosphatase inhibitor cocktail (ASPEN, USA). We used Bicinchoninic acid (BCA) assay (ASPEN, USA) to estimated protein concentration after centrifugation at 13000 rpm for 5 min. Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes. Then, the membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit monoclonal anti-GAPDH antibody (diluted 1:10000, Abcam), rabbit monoclonal anti- p-AMPKα1 antibody (diluted 1:1000, Abcam), rabbit monoclonal anti- PGC-1α antibody (diluted 1:500, Abcam), rabbit monoclonal anti-PPARα antibody (diluted 1:1500, Abcam), rabbit monoclonal anti-FAT antibody (diluted 1:500, Bioss), rabbit monoclonal anti-VLCAD antibody (diluted 1:1000, Abcam) and then with secondary HRP-goat anti-rabbit antibody (diluted 1:10000, ASPEN) for 30 min at room temperature (RT). For loading controls, the membranes were stripped with stripping buffer (ASPEN, USA) for 10 min at room temperature. Antibody binding was detected with the ECL detection reagent (ASPEN, USA). At last, we quantified bands with AlphaEaseFC Software and data are shown as the ratio of total protein to GAPDH normalized to control.

The total proteins from cells were solubilized in RIPA lysis buffer (ASPEN, China) and were loaded into the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to the polyvinylidene fluoride membranes (Millipore, Germany) and blocked with 5% skim milk powder for 1 h. The membranes were then incubated overnight at 4°C with primary antibody as follows: P-IκBα rabbit monoclonal antibody (ab133462, 1:1000, Abcam, Cambridge, UK), cleaved caspase3 rabbit polyclonal antibody (ab49822, 1:1000, Abcam, Cambridge, UK), Ki-67 rabbit monoclonal antibody (ab92742, 1:500, Abcam, Cambridge, UK), GAPDH rabbit polyclonal antibody (ab37168, 1:10000, Abcam, Cambridge, UK), p65 rabbit monoclonal antibody (#8242, 1:2000, CST, USA), cyclinD1 rabbit monoclonal antibody (#2878, 1:1000, CST, USA), IκB-α rabbit polyclonal antibody (18220-1-AP, 1:2000, Proteintech Group, Inc., USA), and MT1X rabbit polyclonal antibody (OACA02206, 1:500, avivasysbio, USA). Then, the membranes were incubated with horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG (AS1107, 1:10000, ASPEN, China) at room temperature for 1 h and were washed with PBS containing 0.1% Tween-20 buffer for 10 min 3 times. The protein bands were visualized with an ECL kit (ASPEN, China), and the band intensity was analyzed using AlphaEaseFC software.